EP0184576A1 - Promotoren für die Expression von fremden Genen in Hefe, Plasmide, die diese Promotoren enthalten, sowie deren Verwendung zur Herstellung von Polypeptiden - Google Patents

Promotoren für die Expression von fremden Genen in Hefe, Plasmide, die diese Promotoren enthalten, sowie deren Verwendung zur Herstellung von Polypeptiden Download PDF

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Publication number
EP0184576A1
EP0184576A1 EP85870171A EP85870171A EP0184576A1 EP 0184576 A1 EP0184576 A1 EP 0184576A1 EP 85870171 A EP85870171 A EP 85870171A EP 85870171 A EP85870171 A EP 85870171A EP 0184576 A1 EP0184576 A1 EP 0184576A1
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plasmid
yeast
promoters
promoter
site
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French (fr)
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EP0184576B1 (de
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Jacques Oberto
John R.N. Davison
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Total Research and Technology Feluy SA
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Labofina SA
Fina Research SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)

Definitions

  • the present invention relates to fragments of yeast DNA comprising a promoter capable of ensuring the expression in yeast of genes coding for polypeptides, generally for heterologous polypeptides. It also relates to vector plasmids comprising said fragments and to yeasts transformed by said plasmids.
  • New genetic engineering techniques permit the expression in various organisms of genes coding for foreign proteins.
  • One example of this, among many others, is the synthesis of human interferons by the yeast Socchoromyces cerevisiae (R.A. Hitzman et al., Science 219, 1983, 620).
  • yeast promoter which is recognized by the yeast RNA polymerase II and causes the synthesis of the corresponding RNA messenger.
  • An object of the present invention is to provide such promoters. Another object is to provide vector plasmids wherein said promoters are fused with heterologous genes so as to ensure the expression in yeast of said heterologous genes into the corresponding polypeptides.
  • the yeasts transformed by said plasmids are a further object of the invention, as are the new polypeptides produced by said yeasts. Still another object is to provide a process for producing polypeptides by growing said transformed yeasts.
  • the isolation of the base promoter of the invention hereinafter called p415, used known methods. It relied on the observation (L. Guarente, Meth. Enzymol. 101, 1983, 181; M.J.Casadaban et al., ibid. 100, 183, 293) that the gene lacZ coding for the ⁇ -golactosidase of E. coli can, under certain conditions, be expressed in the yeast S. cerevisiae and that this expression can be monitored by the blue colour of the yeast colonies when grown on the chromogenic indicator X-gal. This blue colour is due to hydrolytic cleavage of the indicator by ⁇ -galactosidase, and the rate of this cleavage depends on the amount of ⁇ -galactosidase synthesized.
  • the blue colour of the colony depends on the efficiency of ⁇ -galactosidase expression, which in turn depends, among other things, on the force of the yeast promoter used.
  • the strongest may give the blue colour more rapidly, and this can be verified quantitatively by enzyme assay of ⁇ -galactosidase activity.
  • a prerequisite for the isolation of yeast promoters according to the above method is a vector capable of replication and selection in yeast, that contains the lacZ gene of E. coli but does not express this in yeast due to the absence of a suitable promoter.
  • Said vector further has to fulfill the following conditions. It should be capable of replicating in the yeast by means of a sequence recognized by the replication machinery of the yeast. It should be capable of selection and maintenance in yeast due to the presence of a marker gene. It-should be potentially capable of expressing the lacZ gene in yeast when given a suitable promoter. It should comprise a unique restriction site upstream of this gene, for the insertion of small fragments of yeast DNA likely to comprise a promoter.
  • Isolation of the promoter of the present invention with the hereabove described promoter probe vector was carried out as follows. DNA fragments resulting from a partial digest, with the Mbol restriction enzyme of total DNA of S. cerevisiae KL14-4A (G. Foge et al., J. Mol. Biol. 99, 1975, 203) were inserted into the BamHI site of the hereabove described plasmid YEpZ36, and the recombinant DNA thus obtained was used to transform the leu2 strain of S. cerevisiae GRF18.
  • the transformed yeast colonies were screened on X-gal medium for ⁇ -galactosidase production. Several clones appeared positive upon transfer to X-gal medium, and the most active one, as determined by f-galactosidase assays, was selected for the isolation of the promoter of the invention. 1
  • Plasmid DNA from this clone isolated after transfer to E. coli showed a 980 bp insert,that contained an EcoRI site 630 bp before the /3-galactosidase gene.
  • the promoter ensuring transcription of.the ⁇ -galactosidase was found to be associated to this 630 bp fragment, as replacement of the 350 bp fragment preceding the EcoRI site did not affect f-galactosidase activity.
  • the method of construction whereby the 350 bp fragment was deleted is detailed on Figure 2, where the initial 980 bp fragment is referred to as p414.
  • both ends were flanked with unique restriction sites in current use. In this way, it can be moved from one plasmid to another, according to the application envisioned, and downstream insertion of a foreign gene for expression from the promoter is facilitated.
  • promoter p415 as it is included into plasmid YEpZ101 is advantageous for the expression of a gene, such as the lacZ gene of pMC1587 or YEpZ100 ( Figure 4) that lacks an ATG initiator codon, since this is provided by the promoter itself just upstream of the BamHI site.
  • promoter p415 of plasmid YEpZ101 cannot be used as such. It is indeed known that in eucaryotes, yeast included, the first ATG codon of the messenger RNA is normally used.
  • a second ATG introduced would have one of two effects, according to whether it would be in phase or out of phase with the ATG codon of the promoter.
  • a polypeptide would be produced which would be totally different from that coded by the gene.
  • the amino acids corresponding to the codons between the first and the second ATG's would be added to the polypeptide at the amino end.
  • a polypeptide differing from that normally coded by the gene, though perhaps not inactive would be obtained; this is unfavorable since substitutes as close as possible to the natural products are sought in most genetic engineering applications. This situation is especially unfavorable when it is also attempted to make use, as in the examples, of the signal sequence of a natural product to ensure its export outside of the transformed yeast cell.
  • promoter p415 it was necessary to have variants of promoter p415 not comprising the ATG initiator codon, while retaining the immediately downstream restriction site.
  • the following examples describe such deletions from plasmid YEpZ101, starting from site BamHI.
  • variants of promoter p415 were obtained which comprise a BamHI site at various distances from the HindIII end:p415 ⁇ 2, p415 ⁇ 4 and p415 ⁇ 5. It is obvious that the BamHI and HindIII ends in these variants can be replaced by other restriction sites just as in the case of the original p415 promoter.
  • the promoters according to the invention can be used to express foreign genes in yeast if they are correctly associated to the gene to be expressed in a vector plasmid capable of replication into an as high as possible number of copies.
  • This vector should therefore comprise a replication origin recognized by the host cell, and also a marker gene allowing visualisation and selection of the cells which have effectively been transformed by the plasmid.
  • a great number of expression vectors comprising these different elements have been constructed, especially for the transformation of yeasts of the S.cerevisiae species. They generally comprise either the replication origin off the 2-micron plasmid present in most strains of this species, or even an autonomous ARS replication segment of chromosomal origin.
  • a gene .coding for an enzyme involved in the biosynthesis of on essential metabolite, e.g. an amino acid, is generally used as marker.
  • the host cell is then used in a yeast strain which has become by mutation auxotrophic for said metabolite. When growing this strain on a medium free of said metabolite, only those cells transformed by a plasmid carrying the missing gene will be able to grow.
  • Typical examples of such markers are the LEU2 or TRP1 genes coding for on enzyme involved in the biosynthesis of leucine and tryptophane respectively.
  • These expression vectors should also comprise one or preferably several restriction sites where to insert the coding, part of interest and the various elements required for optimizing the expression thereof: promoters, terminators and other control elements.
  • these plasmids often comprise bacterial sequences capable of ensuring their replication and selection in an intermediate bacterial host, e.g. E.coli.
  • shuttle plasmids it may be cited YEp13 (J.R.Broach et al., Gene 8, 1979, 121) pFLI-4 (M.R. Chevallier et al., Gene 11, 1980, 11), and pJDB207 (J.D.Beggs, Alfred Benson Symposium N°16, Munksgaard, Copenhagen 1981, p.383).
  • the plasmid used comprises at least the REP functions of the 2-micron endogenous plasmid sequence. These functions generally give a better stability to the plosmid, especially if the host cell has previously been cured of its 2-micron plasmids (C.P. Hollenberg, Curr.Top.Microbiol. Immunol. 96, 1982, 119; R.M. Walmsley et al., Mol.Gen.Genet., 1983, 261).
  • Classical examples of such vectors are plasmids pJDB219 and pJDB248 (J.D.Beggs, Nature 275, 1978, 104). Another vector of this type is described in the following examples.
  • the transformed cells obtained in these various cases are also part of the invention.
  • the invention also comprises the cells obtained by transforming other species and - genera of yeast with expression vectors of the type hereabove described.
  • other yeasts it may be cited Saccharomycopsis lipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis, etc....
  • transformable host cells from the Saccharomyces genes and preferably from the S.cerevisiae species will be used.
  • transformable strains from this species it may be cited AH22 and GRF18 amongst many others.
  • the DNA fragments which can be expressed in yeast according to this invention may have various origins. They may be procaryotic genes, as the E.coli / 3-galactosidase gene whose expression is described in one of the following examples.
  • the DNA fragment may also be an eucaryotic giene as for as it does not comprise introns as yeast is unable to ensure maturation of the messenger RNA from the transcription of fragmented genes (J.C.Beggs et al., Nature 283, 1980,835). In this case, it is however possible to express the corresponding cDNA, as described herebelow in another example showing chicken lysozyme expressed in yeast. Many other genes can similarly be expressed, whether they are of bacterial, vegetable, animal or human origin. New polypeptides that would thus be expressed are obviously also within the scope of the invention.
  • a yeast strain When a yeast strain has been induced according to the present invention to produce one or another of these polypeptides, it is necessary to multiply it under the conditions most favourable to its growth in order to take advantage of this new property.
  • One skilled in the art will easily determine these conditions according to the characteristics peculiar to the yeast strain used as host. As transformed yeasts have in most cases a more or less important tendency to lose artificially-constructed plasmids, it is advantageous to use a culture medium such as to exert a positive selection pressure on them.
  • the strain is an auxotrophic mutant for one or another essential metabolite and when the vector plasmid used comprises a marker gene capable of restoring the strain prototrophy, e.g.
  • this selection pressure may be exerted by omitting said metabolite from the culture medium.
  • the plasmid comprises as marker a gene capable of conferring to the yeast a more or less marked resistance to a growth inhibitor, e.g. an antibiotic such as G418 (J.Jimenez and J.Davies, Nature 287, 1980, 869) or an herbicide such as diuron (Belgian Patent 899,607 to the Applicant), the selection pressure in favour of the transformed yeast can be applied by growing it in a medium supplemented with this inhibitor.
  • a growth inhibitor e.g. an antibiotic such as G418 (J.Jimenez and J.Davies, Nature 287, 1980, 869) or an herbicide such as diuron (Belgian Patent 899,607 to the Applicant
  • the selection pressure in favour of the transformed yeast can be applied by growing it in a medium supplemented with this inhibitor.
  • Other means exist to obtain the same result and may also be used to practice the invention
  • the gene expressed upon intervention of promoter p415 or one of the variants thereof will preferably be equipped with a leader sequence coding for a signal peptide capable of ensuring the transport of the product through the plasmid membrane of the transformed cell.
  • the separation of the polypeptide formed will indeed be considerably easier, whether it is liberated into the medium from where it will be recovered by classical methods, including adsorption and/or precipitation, or it remains associated with the yeast wall from where it will have to be separated by other methods.
  • Plasmid YEpZ415 the construction of which contributed, as hereabove described, to the isolation of promoter p415 according to the invention, was used to transform S.cerevisiae GRF18(Leu - ,His - ) yeast strain with selection of the clones prototrophic for leucine.
  • promoter p415 according to the invention was flanked by two restriction sites commonly used in genetic engineering (HindIII and BamHI), and used in this form to express the lacZ gene of E.coli in a plasmid called YEpZ101. This operation was carried out in several steps which required the construction of two intermediate plasmids: YEpZ100 and pJ04.
  • YEpZ100 (Figure 4) was constructed from pMC1587 (M.J.Casadaban et al., Methods in Enzymology 100, 1983, 293) and YEpZ415 of which it combines the advantages: it has the beginning of the lacZ gene and the group of restriction sites EcoRI, Smal and BamHI of pMC1587; it has the end of the lacZ gene of YEpZ415, whereby are eliminated the lacy and lacA genes of pMC1587 which would uselessly increase the size of the plasmid; it has the further advantage of keeping the high number of copies characteristic to the plasmids derived from pJDB207, e.g. . YEpZ415 (E.Erhart and C.P.Hollenberg, J.Bacteriol. 156, 1983, 625).
  • 2.3 YEpZ101 was then constructed by combining DNA fragments from three of the plasmids described above, by a process involving three ligation events ( Figure 6).
  • the lacZ gene, the amp R gene, the replication origin in E.coli, the LEU2 gene, and the replication origin in S.cerevisiae all derive from YEpZ100.
  • the promoter p415 is reconstituted intact from two parts : (a) the 5' terminal half from the HindIII-ClaI fragment of YEpZ415, and (b) the 3' proximal half from the ClaI-BomHI fragment from pJ04.
  • the next result of these operations is that the promoter p415 is now bounded by a unique HindIII site upstream and a unique BamHI site downstream, the latter being able to serve for the introduction of foreign genes as described herebelow.
  • plasmid plys ⁇ 9 was available which comprises the complete full cDNA of chicken lysozyme with its own ATG codon as described in Belgian patent 901,223.
  • This cDNA had still to be appropriately combined with a yeast promoter as described for the ⁇ -galactosidase gene in the preceding example.
  • a plasmid such as YEpZ101 comprising promoter p415 which itself comprises an ATG codon.
  • a variant of this plasmid free from this ATG codon was therefore required.
  • the plasmids constructed as described hereabove were then transformed into GRF18 strain (Leu , His ) of the yeast S.cerevisice, followed by selection for clones prototrophic for leucine (Leu + ).
  • lysozyme was evidenced around transformed colonies growing on Petri dishes covered with a lawn of M. lysodeikticus.
  • lysozyme expression was visualised by a transparent halo of bacterial lysis around the colonies.
  • the halo of lysis was greatest with plys ⁇ 49 indicating that this clone was the best producer of lysozyme.
  • This result also showed that the lysozyme is exported from the yeast cell since it must of necessity be extracellular in order to lyse the bacterial indicator.
  • Figure 10 also shows that by operating in the same way with the plys ⁇ 29 clone, no cell lysis could be observed.
  • the sequences responsible for the promoter function should therefore not only be present upstream of the gene to express, but also be suitably positioned with respect to this gene.
  • the vector pJDB207 upon which the lysozyme expression plasmids plys ⁇ 29, plys ⁇ 49 and plys ⁇ 59 (described hereabove) are based does not contain the entire 2-micron yeast plasmid and is consequently dependant on the presence of the endogenous 2-micron plasmid (found in most strains of S.cerevisiae) for its continued maintainance. This leads to such an unstable situation that the pJDB207-type plasmids are frequently lost from the cell (E. Erhaert & C.P.Hollenberg, J. Bacteriol. 156, 1983, 625, and M.Jarayam et al., Cell 34, 1983, 95).
  • the natural 2-micron plasmid is stably inherited. It is indeed known that plasmids constructed in such a way that they contain the entire 2-micron plasmid pJDB219, are more stable than those of the pJDB207-type (C.P. Hollenberg, Curr. Top. Microbiol. Immunol. 96, 1982, 119; R.M. Walmsley et al., Mol. Gen. Genet. 1983, 361). Such plasmids are therefore more useful for long term growth, as for example in industrial fermentations.
  • plasmid YEpB2 ( Figure 11) was used, which was previously made by cloning the entire 2-micron plasmid into pBR322 at their mutually unique PstI sites. Two fragments from YEpB2 and two fragments from plys ⁇ 49 were then combined to give plys50 ( Figure 12).
  • the three 2-micron genes A, B and C are intact and the lysozyme gene is expressed from the p415A4 promoter as in plys ⁇ 49 described in the preceeding example.
  • Lysozyme activity in the supernatant and the lysate from both culture was determined by their ability to lyse M.lysodeikticus cells according to the method of D.Shugar (Biochem. Biophys. Acta, 8, 1952, 302). No activity was detected in a homogenate of the strain transformed by plasmid pJDB207, whereas a lysozyme activity of 35 units per ml of culture could be shown for that of strain GRF18(plys50).

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EP85870171A 1984-12-06 1985-12-05 Promotoren für die Expression von fremden Genen in Hefe, Plasmide, die diese Promotoren enthalten, sowie deren Verwendung zur Herstellung von Polypeptiden Expired - Lifetime EP0184576B1 (de)

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Application Number Priority Date Filing Date Title
AT85870171T ATE54167T1 (de) 1984-12-06 1985-12-05 Promotoren fuer die expression von fremden genen in hefe, plasmide, die diese promotoren enthalten, sowie deren verwendung zur herstellung von polypeptiden.

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Application Number Priority Date Filing Date Title
BE214123 1984-12-06
BE0/214123A BE901222A (fr) 1984-12-06 1984-12-06 Promoteurs assurant l'expression de genes etrangers chez la levure, plasmides comportant ces promoteurs et leurs utilisations pour la production de polypeptides.

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EP0184576A1 true EP0184576A1 (de) 1986-06-11
EP0184576B1 EP0184576B1 (de) 1990-06-27

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EP (1) EP0184576B1 (de)
JP (1) JPH06104065B2 (de)
AT (1) ATE54167T1 (de)
CA (1) CA1280080C (de)
DE (1) DE3578435D1 (de)

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JPH06104065B2 (ja) 1994-12-21
EP0184576B1 (de) 1990-06-27
US4990446A (en) 1991-02-05
CA1280080C (en) 1991-02-12
ATE54167T1 (de) 1990-07-15
DE3578435D1 (de) 1990-08-02
JPS61185190A (ja) 1986-08-18

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